Voltage-gated proton channels (HV1) are phylogenetically diverse, being distributed from unicellular marine life to humans. They have uniquely small unitary conductance but are perfectly selectivity for protons, which is essential considering the low H+ concentration in cells. HV1 channels are voltage activated and their voltage dependence is strongly regulated by the transmembrane pH gradient ( ), so that they extrude acid from cells. The HV1 channel protein is strikingly similar to the voltage-sensing domain of voltage-gated K+ and Na+ ?pH channels. Mammalian HV1 channels are dimers with a conduction pathway through each protomer (incredibly these gate cooperatively!). HV1 functions in humans are diverse, including phagocyte bacteria killing, airway pH regulation, basophil histamine release, sperm maturation, and B lymphocyte responses. Elevated HV1 expression exacerbates chronic lymphocytic leukemia, breast, and colon cancer metastasis, and contributes to brain damage from ischemic stroke. Indeed, recognition of the clinical importance of HV1 channels is rapidly expanding. In contrast, the molecular mechanisms that control HV channel function remain poorly understood. This makes development of rational HV1 channel targeted therapeutic interventions mechanistically blind and thus nearly impossible. Only one crystal structure exists (of a presumed closed state), and it has suspected flaws due to its being a chimera. Thus, the exposed parts of the channel (i.e. those assessable by aqueous therapeutics), especially when open, are very poorly defined. To overcome this knowledge barrier, we will identify clinically pertinent structure-functional attributes of HV1 gating and pH sensing using a combination of state-of-the-art electrophysiology, molecular biology, and protein biochemistry. We will test the following overall hypothesis: The clinically relevant pH sensing, permeation, and gating mechanisms of the H channel are functionally linked via multiple protonatable groups, V1 providing a redundancy that safeguards the essential physiological function of proton channels. By addressing this hypothesis our aims will systematically establish a strong mechanistic foundation that defines possible points of therapeutic intervention. In Aim 1 we will determine the molecular mechanism that couples transmembrane pH gradient and HV1 channel gating. The unique ?pH dependence of gating is the single most important property of the HV1 channel and is central to all known HV1 functions, yet has eluded all efforts to understand its mechanism. Experiments will be guided by three novel mechanistic models that involve charges in the protein and protonation sites. By identifying the correct pH sensing mechanism, critical molecular components can be located. Aim 2 focuses on understanding the gating mechanism of HV1, especially the location and solvent accessibility of residues in closed and open channels. We will study mutants with unique gating properties; model closed, intermediate, and open states using molecular dynamics (MD); and evaluate the role of dimerization in the responses of HV1 in phagocytes, sperm, and other cells. Identifying the key parts of the molecule involved in gating will enable regulating channel opening in health and disease.